1. Field of the Invention
The present invention relates to a culture technology for multipotent undifferentiated cells, such as an ES cell, an adult multipotent stem cell, etc., and particularly relates to a culture substrate and a culture method using this and a cultured cell which forms an undifferentiated 3-dimensional cell block.
2. Description of the Related Art
Damage to a tissue by a disease, aging, an accident, etc. affects human life activity seriously. Regeneration capacity of a human tissue is low. For example, repair of a bone, a cartilage, etc. is carried out by filling a damage portion with an implant, such as an artificial bone.
On the other hand, in a cell culture technique in recent years, due to improvements in development of isolation of an undifferentiated cell from human bone marrow, differentiation and induction into a target tissue cell, a 3-dimensional culture technique, and a scaffold, etc., it is possible to prepare tissues of a skin, a bone, a cartilage, a blood vessel, a cardiac valve, a ligament, etc., from an adult stem cell, and clinical applications have already begun partially.
Thus, in order to obtain a complicated tissue from an undifferentiated cell by way of cell culturing it is necessary to culture the undifferentiated cell represented by an embryonic stem cell whilst maintaining it in an undifferentiated condition, by using a culture substrate having a 3-dimensional structure, to form a 3-dimensional cell block (colony), to supply a cell growth factor and a nutrient to this 3-dimensional cell block, and to differentiate and induce the undifferentiated cell into a target tissue.
In recent years, research of differentiation and induction in the tissue from such an undifferentiated cell has been carried out actively. As an example of this, it is reported that an undifferentiated cell is cultured and proliferated in a porous hydroxyapatite having bio-stability, bio-affinity, and a porosity of 75%, then the porous hydroxyapatite is transplanted in the recipient's bone for therapeutic effects (for example, see Japanese Patent Publication (KOKAI) No. 2002-17846).
Further, in order to culture and maintain the undifferentiated cell represented by the embryonic stem cell etc., a culture substrate is used in which an biopolymer, such as collagen, gelatin, laminin, matrigel, etc., is coated on a plastic petri dish, a feeder cell represented by MEF (murine embryonic fibroblast), is cultured to form a feeder layer onto which an ES cell is seeded (introduced) to be cultured.
It is thought that since calcium phosphate type ceramics, such as a hydroxyapatite, as disclosed in the above-mentioned Japanese Patent Publication No. 2002-17846, is excellent in bio-stability and bio-affinity, it is preferable as a substrate which does not cause immune rejection.
However, Ca2+ ion which is the main components of the hydroxyapatite is important factors when an osteoblast forms a bone. Thus, in the case where the calcium phosphate type ceramics, such as the hydroxyapatite etc., are used as the culture substrate for the undifferentiated cells such as the ES cell etc., it is difficult to keep the cells undifferentiated, because they are differentiated and induced into another type of cells due to the influence of Ca2+ ion etc.
Further, in a method of using the feeder cells, such as the above-mentioned MEF etc., the ES cell may not be separated from other cells completely after culturing. Furthermore, it is possible that in the case where the biopolymer or the feeder cell is used as a substrate for regeneration therapy, there is a possibility that it may be contaminated with a dangerous factor or cause immune rejection, and there is a worry that it may provide adverse effects clinically.
For this reason, research and development has been carried out to culture an ES cell on a culture substrate without using the feeder cells, such as MEF, however a undifferentiated complete 3-dimensional culture block has not formed yet.
Therefore, there is a need for developing a culture substrate which can grow a human ES cell etc. efficiently, avoid a dangerous factor and immune rejection, and culture an undifferentiated 3-dimensional cell block.